Is There a Significant Role for Lipid Peroxidation in the Causation of Malignancy and for Antioxidants in Cancer Prevention?1

a-Tocopherol (a-T) uptake and its relationship to cell proliferation and lipid peroxidation was studied in a baby hamster kidney cell line (BHK21/C13) and its polyoma virus-transformed malignant counterpart i IÃŽIIK21/PyY cells). The principal findings were as follows, (a) The level of lipid peroxidation judged by malondialdehyde (MDA) measurement by HPLC, was higher in the transformed cells than in the nontransformed cells. Oxidative stress by 374 mM Fe3+/10 HIMADP caused a significant increase in the level of MDA of a similar magnitude in both cell types. Addition of 7, 14, and 21 mM a-T caused no diminution of the MDA level in the unstressed cells and abolished the increase in MDA seen in the stressed cells, i /• i The endogenous level of a-T in the transformed cells was lower than in the nontransformed cells and all of the measurable a-T in these cells was destroyed by the oxidative stress. Supplementation of the cells with a-T caused an increase in the level of a-T that was proportional to the level of inclusion of a-T in the medium, (r) (Growth was stimulated by 7 and 14 HIMa-T but not by the higher levels of inclusion in the medium. The growth stimulation was much larger in the transformed cells (163% of growth in the unsupplemented medium) than in the nontransformed cells (120%). u/i These results demonstrate that, in this cell system, the growth-stimulating ability of a-T is unrelated to the ability of a-T to control lipid peroxidation and that the level of peroxidation is increased in the malignant state. The difference between the findings reported here and earlier work showing increased levels of a-T and decreased levels of peroxidation in transformed malignant cells is discussed and possible explanations for it are advanced. Relationship between Human EpidemiolÃ3gica! Studies and Biochemical Studies The role of oxidative processes involving free radicals in the carcinogenic process is now well established and accepted by most authorities (1). Antioxidants which may be acting as free radical scavengers inhibit this process and it is therefore likely that dietary antioxidants such as vitamin C. vitamin E, and ß-carotenemay be important in inhibiting the cancer process in vivo (2, 3). In a recent review, Block et al. (2) examined 200 reports of studies of the relationship between fruit and vegetable intake and cancers of the lung, colon, breast, cervix, esophagus, oral cavity stomach, bladder, pancreas, and ovary; implicit in these studies was the assumption that a large part of the protective ability of fruit and vegetables was derived from their content of the antioxidant nutrients mentioned above. A statistically significant protective effect of fruit and vegeta ble consumption was found in 128 of 156 dietary studies in which results were expressed in terms of the relative risk. For most cancer sites it was found that individuals with a low fruit and vegetable intake experience about twice the risk of disease compared to those with high intake. For lung cancer particularly strong correlations were found in 24 of 25 studies, and fruit in particular was significantly protective in 28 of 29 studies of cancers of the esophagus, oral cavity, and larynx. Strong correlations were also found for the other cancer sites and 1 Presented al Ihe 4th International Conference on Anticarcinogenesis & Radiation Protection. April 1X-23. lutJ3. Baltimore, MD. Financial support was from Ihe Associ ation for International Cancer Research, the Association of Commonweallh Universities, and the Medical Research Council. 2 To whom requests for reprints should he addressed. Block et al. concluded that "major public health benefits could be achieved by substantially increasing consumption of these foods." Studies at the fundamental biochemical level have also been carried out and have been directed to an investigation of the role of free radicals in the carcinogenic process and of the inhibition of this process by antioxidants. Free radicals have been implicated in both the initiation and promotion stages of carcinogenesis. The body has a range of scavenging systems located extracellularly and intracellularly to combat excessive production of free radicals (1). Examination of certain malignant cell types showed (4, 5) decreased susceptibility to lipid peroxidation compared with their nonmalignant counterparts which appeared to be in direct relation to their degree of dedifferentiation (6). The mechanisms of this resistance are unclear since the activities of some intracellular antioxidant enzymes are decreased in cancer cells (7); the activities of the Superoxide dismutases, both cytoplasmic and mitochondria!, are usually found to be lower and that of catalase almost always lower. The levels of the selenium-contain ing enzyme glutathione peroxidase, and of glutathione reducÃ-ase,are highly variable; the activity of the peroxidase is generally higher and that of the reducÃ-ase lower, even though Ihe aclivily of glucose-6phosphale dehydrogenase, Ihe supplier of reducing equivalents for glutalhione reducÃ-ase,is increased. In regeneraling rat liver Ihe periodic bursls of DNA replicalion, as cells progress through a number of division cycles, are accompanied by corresponding depressions in the susceptibility of cell components to lipid peroxidation (8). Levels of a-tocopherol increase in relation to Ihese periods of DNA synthesis, and it may be that a-tocopherol is a major conlribulor to (he decreased peroxidalive susceplibility during periods of DNA replicalion in regeneraling ral liver. Il is not clear at present whelher Ihe observed increased resislance to lipid peroxida tion is a property of malignant cells, or whether it is simply a feature of rapidly dividing cells, which is encountered in regenerating rat liver or in rapidly proliferating tumors. Thus, the increased resistance to peroxidation in transformed cells seen in these experiments is para doxical in the sense that the preventive effect of antioxidants seen in human epidemiological studies would be expecled to be relaled to the abilily of Ihese antioxidants to cause a decrease in oxidative damage that may contribute to some stage of the carcinogenic process. A criticism of earlier experiments that demonstrate a lower level of peroxidation in cancer cells is that the controls used in them were for the most part different from Ihe cancer cells and that, therefore, direct comparison between them may not be valid. For example, the studies of Slaler's group (5, 8) of Novikoff and Yoshida tumors used as comparative conlrols the normal hepatocyles of the animals in which the tumors were implanted. These cells were therefore genetically and phenotypically different from the implanted tumor cells. The present study was designed to attempt to overcome this criticism of earlier work by investigating the relative differences in a-tocopherol uptake and its effect in terms of cell growth in an immortal cell line of baby hamster kidney fibroblasts (BHK-21/C13) and in the same baby hamster kidney fibroblasts transformed to the malignant slate using polyoma, a DNA tumor virus (BHK-21/PyY cells). The effect on the cells of the application of an exogenous stress by Irealmenl with Fe3+/ADP was also investigated. A report of Ihe delails of ihis work